Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3^−/−; 2. C7^−/−; and 3. Cd59ab^−/−) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3^−/−) and MAC formation (in C3^−/−. C7^−/−, and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab^−/−) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3^−/−, C7^−/− mice, and Cd59ab^−/− mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19.

Within arterial plaque, HIV infection creates a state of inflammation and immune activation, triggering NLRP3/caspase-1 inflammasome, tissue damage, and monocyte/macrophage infiltration. Previously, we documented that caspase-1 activation in myeloid cells was linked with HIV-associated atherosclerosis in mice and people with HIV. Here, we mechanistically examined the direct effect of caspase-1 on HIV-associated atherosclerosis. Caspase-1-deficient (Casp-1^−/−) mice were crossed with HIV-1 transgenic (Tg26^+/−) mice with an atherogenic ApoE-deficient (ApoE^−/−) background to create global caspase-1-deficient mice (Tg26^+/−/ApoE^−/−/Casp-1^−/−). Caspase-1-sufficient (Tg26^+/−/ApoE^−/−/Casp-1^+/+) mice served as the controls. Next, we created chimeric hematopoietic cell-deficient mice by reconstituting irradiated ApoE^−/− mice with bone marrow cells transplanted from Tg26^+/−/ApoE^−/−/Casp-1^−/− (BMT Casp-1^−/−) or Tg26^+/−/ApoE^−/−/Casp-1^+/+ (BMT Casp-1^+/+) mice. Global caspase-1 knockout in mice suppressed plaque deposition in the thoracic aorta, serum IL-18 levels, and ex vivo foam cell formation. The deficiency of caspase-1 in hematopoietic cells resulted in reduced atherosclerotic plaque burden in the whole aorta and aortic root, which was associated with reduced macrophage infiltration. Transcriptomic analyses of peripheral mononuclear cells and splenocytes indicated that caspase-1 deficiency inhibited caspase-1 pathway-related genes. These results document the critical atherogenic role of caspase-1 in chronic HIV infection and highlight the implication of this pathway and peripheral immune activation in HIV-associated atherosclerosis.

Pneumocystis pneumonia (PCP) is a serious fungal lung infection caused by Pneumocystis jirovecii in immunosuppressed individuals. The lack of viable in vitro/ex vivo PCP models has greatly hindered the progress in studying the biology of these fungi, the host/pathogen interactions, as well as antifungal susceptibility testing. In this study, we show the utility of precision-cut lung slices (PCLS) to support the survival of Pneumocystis murina in vitro . We cultured PCLS tissue derived from wild type and immunocompromised mice with a P. murina inoculum in submerged or air-liquid interface models for up to 14 days. We isolated total RNA from the cultured lung tissues at days 0, 3, 7, 10, and 14 and analyzed for the expression of host lung genes and P. murina genes ( Gsc1 for asci and Sp for trophs) by real-time quantitative PCR. When cultured in media alone, P. murina died gradually within a few days. However, when cultured on PCLS, both the troph and ascus forms survived throughout the incubation period of 2 weeks. Moreover, immunohistochemistry staining of P. murina inoculated PCLS sections using polyclonal anti- Pneumocystis sera and showed evidence of fungal aggregation and possible biofilm formation. Additionally, in vitro (PCLS) antibiotic susceptibility testing using commonly used antifungal drugs confirmed successful targeting of the troph and ascus forms by trimethoprim sulfamethoxazole and the ascus form by echinocandins. IMPORTANCE: Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

Single-cell RNA-seq has been used to characterize human COVID-19. To determine if preclinical models successfully mimic the cell-intrinsic and -extrinsic effects of severe disease, we conducted a meta-analysis of single-cell data across five model species. To assess whether dissemination of viral RNA in lung cells tracks pathology and results in cell‐intrinsic and ‐extrinsic transcriptomic changes in COVID-19. We conducted a meta-analysis by analyzing six publicly available, scRNA-seq data sets. We used dual mapping (host and virus) and differential gene expression analyses to compare viral ⁺ and viral ⁻ cell populations. We conducted a principal component analysis to identify successful models of human COVID-19. We found expression of viral RNA in many non-epithelial cell types. Fibroblasts, macrophages, and endothelial cells exhibit clear evidence of viral-intrinsic and -extrinsic effects on host gene expression. Using viral RNA expression, we found that K18-hACE2 mice most closely modeled severe human COVID-19, followed by hamsters. Ferrets and macaques are poor models of human disease due to the low presence of viral RNA. Moreover, we found that increased transcripts of certain key inflammatory genes such as IL1B, IL18 , and CXCL10 are not restricted to virally infected cells, suggesting these genes are regulated in a paracrine or autocrine fashion. These data affirm widespread dissemination of viral RNA in the lung, which may be key in the pathogenesis of severe COVID-19 and demonstrate ferrets and Rhesus macaques are poor models of human COVID-19. IMPORTANCE: We conducted a high-resolution meta-analysis of scRNA-seq data from humans and five animal models of COVID-19. This study reports viral RNA dissemination in several cell types in human data as well as in some of the pre-clinical models. Using this metric, the K18-hACE2 mouse model, followed by the hamster model, most closely resembled human COVID-19. We observed clear evidence of viral-intrinsic effects within cells (e.g., IRF5 expression) as well as viral-extrinsic cytokine modulation (e.g., IL1B, IL18, CXCL10 ). We observed proinflammatory chemokine expression in cells devoid of viral RNA expression, suggesting autocrine/paracrine interferon regulation. This report serves as a resource-synthesizing data from COVID-19 humans and animal models and suggesting improvements for relevant pre-clinical models that may aid future diagnostic and therapeutic development projects.

Pneumocystis jirovecii is the most common cause of fungal pneumonia in children under the age of 2 years. However, the inability to culture and propagate this organism has hampered the acquisition of a fungal genome as well as the development of recombinant antigens to conduct seroprevalence studies. In this study, we performed proteomics on Pneumocystis-infected mice and used the recent P. murina and P. jirovecii genomes to prioritize antigens for recombinant protein expression. We focused on a fungal glucanase due to its conservation among fungal species. We found evidence of maternal IgG to this antigen, followed by a nadir in pediatric samples between 1 and 3 months of age, followed by an increase in prevalence over time consistent with the known epidemiology of Pneumocystis exposure. Moreover, there was a strong concordance of anti-glucanase responses and IgG against another Pneumocystis antigen, PNEG_01454. Taken together, these antigens may be useful tools for Pneumocystis seroprevalence and seroconversion studies.